Study Of The Roles And Mechanisms Of Vitamin D₃ Up-regulated Protein 1(VDUP1) In Oxidative Stress In Asthma

BACKGROUND AND AIM:Bronchial asthma is a chronic respiratory disease characterized by chronic airway inflammation,airway hyperresponsiveness,airway remolding,variable airway obstruction,which has great impact on human's health.Its chronicity,instability and recurrent episodes of airflow limitation reflect that it results from complicated mechanisms and it is difficult to cure asthma.Asthma behaves as a spectrum of disorders initiated at different stages throughout life by a range of environmental factors interacting with a susceptible genetic background.There are several closely related phenotypes in asthma,of which chronic inflammation depends on regulation by many genes.Recently strong evidences show that gene and environment factors coordinately play a critical role in incidence and development of asthma.Oxidative stress has been involved in nature history of asthma,airway inflammation,airway hyper-responsiveness,increased airway microvascular and epithelial hyperpermeability and tissue injury and remodeling.As inflammation is often associated with an increased generation of reactive oxygen species(ROS),and the biochemical environment in the asthmatic airways is favourable for free radical mediated reactions,it is rational to surmise that an oxidant stress could be mechanistically important in asthma.Also the presence of oxidative stress has important consequences for the severity,and treatment of asthma.But the precise role and clinical siganifiance of oxidative stress in asthma remain to be further explored.Recently the anti-oxidant treatment,such as N-acetylcysteine has been extensive used in many lung diseases,but its use in asthma is rather limited,which may related to the labile nature of ROS,their numerous cellular effects as well as the role of ROS in pathobiology and physiology of asthma remain unknown.It is very helpful for us to futher explore the mechanism of oxidative stress in asthma and find the special factor of oxidative stress for asthmaA long-recognized property of airway epithelial cells(AEC) is their function as a complex physical barrier that defends against exposures to potentially harmful inhaled substances and microbial pathogens.The defect theory of AEC in asthma has been extensively received in recent ten years.It is now clear that AEC also play crucial roles in initiating and augmenting airway host defense mechanisms. Activation of the innate immune response secondarily induces recruitment of immune cells into epithelium to initiate adaptive immunity.By contrast,prolonged and/or robust epithelial activation and reactivation of epithelial-mesenchymal trophic unit (EMTU) can result in the release of large quantities of proinflammatory cytokines, growth factors and chemokines that attract inflammatory cells and that initiate and sustain airway inflammatory diseases such as asthma.The role of the airway epithelium in the pathogenesis of airway inflammatory diseases has been extensively studied and is well established.the AEC from asthma patients was more suspectable to oxidative stress,more fragile to apoptosis,which indicates that the redox state in AEC may play an initiative and critical role in asthma.Our research focus on VDUP1,which has been indentified to be related to eosinophils activation and pulmonary function in our previous studies.VDUP1 as a early response gene,has many biological function,involing in oxidative stress, mechanical stress signal transduction,cell growth and differentiation,lipids metabolism and mucous immunity,which closely related to many complicated diseases such as diabetes mellitus,tumor,and Cardiovascular Diseases.VDUP1 is an important inhibitor of TRX which involves one of the two paramout antioxidant systems called GSH and TRX systems.So this study further explored the roles of VDUP1 in oxidative stress of asthma, and its molecular mechanisms in AEC oxidative stress,which may help to elucidate the basic characteristics of asthma and lay some theoretic foundation for future targeting therapy for asthma oxidative stress.METHODS:1.To investigate the expression of VDUP1 in the lungs of the asthmatic mice and the effect of N-acetylcysteine(NAC.Fifteen female BALB/c mice were randomly divided into control group,asthma group and NAC treatment group(n=5 per group).VDUP1/β-actin gene fragments were amplified simultaneously by RT-PCR for the total lung tissue RNA,VDUP1 protein expression and distribution was detected by immunohistochemical method.2.To investigate the effect of hydrogen dioxide(H₂O₂) on the expression and distribution of VDUP1 and TRX in normal human bronchious epithelial cell line (HBE135-E6E7,HBE) after estabilished the fatal and non-fatal HBE research mode induced by oxidative stress.Methyltetrazolium(MTT) assay was used to assess the HBE cell viability under different concentration of H₂O₂ such as 50μM,200μM and 600μM.VDUP1/β-actin gene fragments were amplified simultaneously by RT-PCR for the total HBE RNA,VDUP1 protein expression and distribution was detected by immunofluorescence technique.The expressions of VDUP1 at both mRNA and protein levels are compared between different groups,immunofluorescence double staining sample for laser confocal microscope assay was used to detect the expression and distribution of VDUP1/TRX.Then further analysised differentital response and synergistic effect of VDUP1 and TRX in HBE treated with H₂O₂,and decided the relation between the expression the distribution of VDUP1 /TRX and cell apoptosis detected by Hoechests33342 assay.3.To explore the effect on cell growth,apoptosis and oxidative stress torlance after siRNA VDUP1.After finished siRNA VDUP1 plasmid construction,immunofluorescence microscope assay was used to evulate the transfection effency.The siRNA VDUP1 effency was detected by RT-PCR and Western blot.MTT assay was used to assess the HBE Cell viability,different concentration of H₂O₂ such as 50μM,200μM, 600μM,1000μM were used to stimulate HBE after siRNA VDUP1,Hoechest assay was used to evulate cell apoptosis.4.To investigate the role and mechanicsm of VDUP1 in H₂O₂ induced the expression VEGF in HBE in this processs.Different concentration of H₂O₂(50μM,200μM,600μM) were used to treat with HBE under the serm-free conditions.VEGF/β-actin gene fragments were amplified simultaneously by RT-PCR for the total HBE RNA,VDUP1 protein expression was detected by ELISA technique.The expressions of VEGF at protein levels are compared among normal groups,50μM H₂O₂,and Ly294002(10μM) pretreatment then 50μM H₂O₂ treatment.After cofirming that H₂O₂ can induce expression VEGF in HBE completely depend on PI₃K pathway.Immunofluorescence double staining sample for laser confocal microscope assay was used to detect the effect of 50μM H₂O₂ on the expression and distribution of VDUP1/TRX and VDUP1/Jabl after 45min Ly294002(10μM) pretreatment.ELISA assay was used to detect the effect of siRNA VDUP1 on protein expression of VEGF after 50μM H₂O₂ treatment.And further investigated the roles and mechanicsms of VDUP1 in H₂O₂ induced the expression VEGF in HBE in this processs.Statistical calculations were performed using SPSS 11.5 software.All data are expressed as mean±SEM.An analysis of variance(ANOVA).factorial designs and LSD methods were used to determine differences between all experimental groups.RESULTS:1.The expression of VDUP1 significantly increased in asthma group and NAC treated group.VDUP1 was distributed mainly in the epithelial cells of bronchi and alveoli,and some of mesenchymal cells.VDUP1/β-actin ratio in control,asthma and NAC treatment group were 0.633±0.055,0.922±0.052,1.07±0.076 respectively.The ratio significantly increased in asthma model group in comparison with control group,which also occur in NAC treatment group(n=6,P＜0.001).The expression also significantly increased in NAC treatment group comparing with asthmatic group(n=6,P＜0.001).the density of VDUP1 protein in control,asthma and NAC group were 66.7±2.9,77.9±3.2,98.3±7.3, the density significantly increased in asthmatic model group(n=5,P=0.004) and NAC treatment group(n=5,P＜0.001) comparing with control group,there was also significant differentce between asthma group and NAC treatment group.VDUP1 was distributed mainly in the cytoplasm of the epithelial cells of bronchi and alveoli in control group,also can be seen in nucleus and membrane2.H₂O₂ can significantly increase the expression of VDUP1 and TRX,the concentration to induce VDUP1 expression is lower than to induce TRX expression. H₂O₂ can induce translocation of VDUP1 and TRX to cell membrance and nucleus. The expression of the two proteins positively correlated with HBE apoptosis.200μM H₂O₂ did not influence the Cell viability;VDUP1/β-actin ratio significantly increased in 50μM H₂O₂ treatment group(n=6,P=0.008),in 200μM H₂O₂ treatment group(n=6,P=0.012) and in 600μM H₂O₂ treatment group(n=6, P＜0.001) in comparison with control group.The expression TRX/β-actin ratio only significantly increased in 600μM H₂O₂ treatment group comparing with control group(n=6,P=0.040).the fluorescence density of VDUP1 significantly increased in 50μM H₂O₂ treatment group,and TRX significantly increased in 200μM H₂O₂ treatment group.VDUP1 and TRX was distributed mainly in the cytoplasm of the epithelial cells in control group,but both of them can be translocated to cell membrance,some of them to nucleus after treated with 50μM H₂O₂,and VDUP1 and TRX always colocalizate.The fluorescence density of VDUP1 and TRX increased significantly under the conditions of cell apoptosis.3.siRNA VDUP1 did't influence growth of normal HBE significantly,but can reduced cell apoptosis under higher concentration of H₂O₂.The transfection effency of siRNA VDUP1 was about 80%by immunofluorescence microscope assay.The siRNA VDUP 1 effency was about 60%. There is no significant difference on the cell growth among normal control group, negative group and siRNA VDUP1 group by MTT assay,neither to cell apoptosis after 24h coculture with siRNA VDUP1.at the concentration of 50μM,200μM H₂O₂, siRNA VDUP1 had no effect on HBE apoptosis.The apoptosis rate at the concentration of 400μM,600μM and 1000μM H₂O₂ before and after VDUP1 siRNA were(22.333±2.081,14.333±2.082,n=3,P=0.009),(36.000±1.826,25.000±3.000, n=3,P=0.002),(44.333±4.041,35.000±3.000,n=3,P=0.033) respectively.In these H₂O₂ concentrations VDUP1 siRNA could reduce the apoptosis rate of HBE.4,the role and mechanicsm of VDUP1 in H₂O₂ induced the expression VEGF in HBE in this processs.â‘ 50μM H₂O₂ can significantly increased the expression VEGF,and Ly294002(10μM) can completely inhibit the effect.HBE135-E6E7 constitively expresses two gene type VEGF₁₈₉ and VEGF₁₆₅. VEGF₁₆₅/β-actin mRNA relative expression was 0.379±0.044,0.791±0.042, 0.585±0.133,0.720±0.0213 treated with 50μM,200μM and 600μM H₂O₂ respectively.VEGF₁₈₉/β-actin mRNA relative expression was 0.193±0.018, 0.270±0.012,0.205±0.074,0.302±0.035 treated with H₂O₂.VEGF165/β-actin and VEGF189/β-actin ratio significantly increased in all three H₂O₂ treatment groups in comparison with control group,the protein expression of VEGF in normal,50μM H₂O₂ treatment group,and Ly294002 pretreatment group was 591.5±9.5 pg/ml, 768.9±21.3 pg/ml,489.3±10.9 pg/ml,respectively,it significantly increased in 50μM H₂O₂(n=4,P＜0.001),and pretreatment of Ly294002 significantly decreased VEGF expression(n=4,P＜0.001),even has significant differentce between normal control and Ly294002 pretreatment group.â‘¡Ly294002 didn't have effect on the expression of VDUP1,TRX and Jab1 and collocation of VDUP1/TRX and VDUP1/Jab1,but can reduce partly H₂O₂ induced VDUP1 membrane translocation.Ly294002 pretreatment has no significant influence on VDUP1,TRX expression dectected by immunofluorescence confocal microscope.Ly294002 pretreatment can reduce H₂O₂ induced VDUP1 membrane translocation,but had no effect on TRX distribution and collocation of TRX and VDUP1.Jab1 normally distributed in the cytoplasm and nuclear,without collocation with VDUPl.after pretreatment of H₂O₂ for 24h,Jab1 can translocate to nuclear or around nuclear membrane,collocation of VDUP1 and Jab1 can be seen in nuclear,and Ly294002 didn't change this situation.â‘¢siRNA VDUP1 can increase VEGF expression slightly,but without statistical differentce.VEGF in supernatant in control,50μM H₂O₂ treatment group,negative control transfection before 50μM H₂O₂ and siRNA VDUP1 transfection before 50μM H₂O₂ was 587.1±12.1 pg/ml,740.1±12.8 pg/ml,748.9±22.5 pg/ml and 759.8±8.4 pg/ml. There were significant difference between control and H₂O₂ treatment group(n=4, P＜0.001),siRNA VDUP1 before 50μM H₂O₂ treatment can increase VEGF concentration slightly,but there was no statistical difference(n=4,P=0.083).CONCLUSION:1.VDUP1 may play a role in the pathogenesis of asthma,which is related to oxidative stress of asthma.NAC may be a new anti-oxidative biomarker in asthma.2.VDUP1 may play a role in oxidiative stress and its tolerance in HBE. VDUP1/TRX may work collaborately in the process of oxidative stress in epithelial cells,which needs to be further studied.3.H₂O₂ can induced expression of VEGF in HBE completely depended on PI₃K pathway,which took part in the the process of VDUP1 translocation.VDUP1,TRX and Jab1 may work collaborately in the process of oxidative stress in epithelial cells. It remains to be further explored whether VDUP1 plays a role in H₂O₂ induced VEGF expression and what its precise mechanism is during this processs.